The cell cycle and the genetic alterations that drive tumorigenesis are inextricably linked. Examples include the amplification of cyclin and cyclin dependent kinase (CDK) genes, the phosphorylation of Rb by CDK'S, the control of the CDK inhibitor p21.sup.WAF1 by p53, and the tumor suppressor activity of the CDK inhibitor p16 (reviewed by Sherr, 1994, Hunter and Pines, 1994).
A turning point in cell cycle research was realized with the discovery that cyclin complexes had different constitutions in transformed and nontransformed cells (Xiong et al., 16992, Xiong et al., 1993). In particular, the complexes in the non-transformed cells were associated with small proteins different from those found in transformed derivatives. A variety of approaches subsequently determined that these small proteins not only bound to the cyclin complexes, but were potent inhibitors of the associated CDK's (p21: Harper et al., 1993, Xiong et al., 1993b, Gu et al., 1993, p16: Serrano et al., 1993, p15: Hannon and Beach, 31994, p18: Guan et al., 1994, p27: Polyak et al, 1994, Toyoshima and Hunter, 1994).
Five CDK inhibitors have thus far been identified. The first to be cloned, p21.sup.WAF1 (also known as CIP1, SDI1, MDA6, CAP20), is encoded by a gene on chromosome 6p and can be directly regulated by p53 (El-Deiry et al., 1993). It inhibits a broad range of cyclin-CDK complexes, and may be involved in cellular senescence as well as in neoplasia (Noda et al., 1994). In vitro, p21.sup.WAF1 also complexes with the proliferating cell nuclear antigen (PCNA), resulting in an inhibition of DNA replication (Li et al., 1994, Flores-Rozas et al., 1994). A related gene, p27.sup.KIP1, is located on chromosome 12p and may play a role in a subset of leukemias (Bullrich et al., 1995, Pietenpol et al., 1995, Ponce-Castaneda et al., 1995). The p16, p15 and p18 genes are unrelated to the p21/p27 family, and have a more selective inhibitory activity, affecting only CDK4 and 6. The p16 and p15 genes are homozygously deleted in many human cancers, and p16 mutations result in familial melanoma (Kamb et al., 1994 a,b, Nobori et al., 1994, Hussussian et al., 1994, Jen et al., 1994).
Although cell culture studies have been used to initially study these CDK inhibitors, such studies fail to provide insight into their regulation in intact animals. There is a continuing need in the art for methods and reagents for diagnosing and treating cancer patients. An understanding of in vivo regulation can lead to new diagnostic and therapeutic tools.